1. Field of the Invention
The present invention is directed to a microscopy device, in particular a microscopy device for the imaging acquisition of fluorescent light.
2. Description of Related Art
It is known in the field of microscopy to use fluorescent light when generating two-dimensional or three-dimensional images of an object to be examined, e.g., a biological sample. In so doing, the fluorescence intensity in particular can be detected. Further, it is possible to use the individual fluorescence lifetimes of fluorophores to generate contrasts. This latter procedure is known as FLIM (Fluorescence Lifetime Imaging Microscopy). The fluorescence lifetime of a fluorophore that can be deliberately introduced in an object to be examined or is a natural component of the object to be examined is largely dependent on the molecular environment of the fluorophore and, therefore, can be used to show the environmental parameters of a fluorophore within the framework of an imaging method. These environmental parameters can include, for example, the fluorophore concentration, pH, temperature, or viscosity. Further, the detection of fluorescent light can be used in analyzing energy transfer processes, e.g., by means of a FRET (Fluorescent Resonance Energy Transfer) mechanism so that it is possible, for example, to obtain information concerning the bonding or convolution characteristics of proteins.
Basically, there are two different known methods for imaging fluorescence lifetime microscopy. A first type of method works in the frequency domain. In this case, an excitation light source which is time-modulated at a frequency in the kilohertz range to the gigahertz range is used. The fluorescent light emitted by the object to be examined in response to the excitation light is acquired in a phase-sensitive manner so that, e.g., a mean fluorescence lifetime can be derived from the phase shift of the acquired fluorescent light. A second type of method works in the time domain. In this case, a pulsed laser with a pulse duration in the femtosecond range to the picosecond range is used to generate the excitation light, and the fluorescence decay curve is acquired at every image point. A time-correlated photon counting can be used in this case, for example.
Microscopy devices suitable for imaging fluorescence lifetime microscopy methods working in the frequency domain are known for raster scanning microscopy methods, i.e., having a single-channel detector, and for widefield microscopy methods, i.e., in conjunction with cameras. However, the problem with raster scanning methods generally is that they are relatively time-consuming with typical image recording times in the range of seconds. Widefield methods typically employ time window-controlled image intensifiers, known as gated image intensifiers, in which a multichannel plate (MCP) whose voltage can be modulated is arranged in front of a CCD (Charge Coupled Device) camera. In this way, time resolutions in the range of 100 ps can be achieved, which is sufficient for many applications. Typically, at least three image recordings are required to determine the phase shift. In multiexponential decay processes, the quantity of required image recordings increases further. This results in problems with respect to the time expended on measurements. Further, problems can arise with respect to possible bleaching processes in the fluorophores. Another problem occurring in the known methods working in the frequency domain is the impossibility of an efficient discrimination of the excitation light or autofluorescence light of the object to be examined with respect to the fluorescence radiation of interest. Signal components of excitation light or autofluorescence light in the detected emission radiation typically lead to a falsification of the determined mean fluorescence lifetime and should therefore be avoided as far as possible.
Microscopy devices for imaging fluorescence lifetime microscopy methods working in the time domain are based, for example, on the combination of confocal or multiphoton laser scanning microscopes and single photon counting and, in this case, can also afford the possibility of generating optical sections. In so doing, the arrival time of individual fluorescence photons relative to a pulse of the excitation light are measured by fast TDC (Time-to-Digital Converter) electronics. Light sources with pulse durations in the femtosecond range to the picosecond range are used for this purpose. The achievable time resolutions are in the range of a few picoseconds. Further, there are known microscopy devices for imaging fluorescence lifetime microscopy methods working in the time domain which use widefield detection and offer the possibility of generating optical sections. A microscopy device of this kind is described, for example, in “High speed optically sectioned fluorescence lifetime imaging permits study of life cell signaling events”, D. M. Grant et al., Optics Express, Vol. 15, No. 24, 15656 (2007). The objects to be examined are excited focally and afocally in this case, although only the focal excitation is actually used, so that there is a problem with excessive bleaching of fluorophores.
Further, the use of SPIM (Selective Plane Illumination Microscopy) is known in connection with fluorescence microscopy methods. This technique is described, for example, in “Optical Sectioning Deep Inside Live Embryos by Selective Plane Illumination Microscopy”, H. K. Stelzer et al., Science 305, 1007 (2004), in “Resolution enhancement in a light-sheet-based microscope (SPIM)”, H. K. Stelzer, et al., Optics Letters, Vol. 31, No. 10, 1477 (2006), and in DE 102 57 423 A1, and WO 2004/0530558 A1
Similar to confocal laser scanning microscopy, SPIM allows the three-dimensional recording of objects in the form of optical sections, but in the framework of a widefield technique. In contrast to fluorescence microscopy using incident light or transmitted light, the fluorophores in the object to be examined are excited by laser light in the form of a light sheet in this case. The use of SPIM in imaging fluorescence lifetime measurements is described in “4D Fluorescence Lifetime Imaging Using Selective Plane Illumination Microscopy”, K. Greger et al., Abstract Book, Focus on Microscopy (Jena, 2005), page 225. A CW (Continuous Wave) laser in the form of an Ar-Ion laser whose output radiation is modulated by a downstream acousto-optical modulator is used in this case.